1. Field of the Invention
The present invention relates generally to molecular biology and molecular genetics and more specifically to mammalian artificial chromosomes.
2. Background Information
The ability to clone and express nucleic acid molecules has resulted in the identification of numerous genes and gene products. As a result of the identification of various genes, molecular differences between normal and diseased conditions are beginning to be recognized. For example, in various disease conditions such as Duchenne muscular dystrophy (DMD), cystic fibrosis and some forms of cancer, mutations in particular genes appear to be the basis for the underlying pathology. In DMD, for example, a mutation in the dystrophin gene can result in the formation of only part of the dystrophin protein, which functions abnormally and contributes to the characteristic signs and symptoms of DMD.
The identification of a molecular defect as the cause of a particular disease suggests possible approaches for ameliorating the disease at the molecular level. Gene therapy, in particular, holds the promise of correcting a pathology such as DMD by introducing a normal dystrophin gene into the muscle cells of an individual suffering from DMD. Unfortunately, the specific molecular defect has been identified in only a handful of diseases. In addition, some genes such as the dystrophin gene contain over one million base pairs and, therefore, are too large to be conveniently transferred from one cell into another using currently available technology.
It has been proposed that the identification of every gene in the human genome will provide insight into the mechanisms responsible for many diseases. Thus, the Human Genome Project was initiated to develop a linkage map for each of the twenty-three pairs of human chromosomes and, ultimately, to obtain the nucleic acid sequence of the entire human genome. However, a structural description, alone, of the human genome is not likely to be sufficient to allow, for example, an understanding of the mechanisms of gene regulation, which can depend on DNA regulatory elements that are located thousands of base pairs or more from the regulated genes.
Currently available mammalian vectors such as retroviral vectors can harbor, at best, DNA fragments containing up to about ten thousand nucleotides. In comparison, yeast vectors such as yeast artificial chromosomes (YACs) can harbor DNA fragments having a few hundred thousand nucleotides. However, such YAC vectors are not stable in mammalian cells and, therefore, cannot be used, for example, as vectors for gene therapy, which, ideally, would be stably maintained in a cell from generation to generation and would express a predictable amount of a gene product. Thus, a need exists for vectors that can contain large fragments of DNA and that are stably maintained in mammalian cells. The present invention satisfies this need and provides related advantages as well.